1. Field of the Invention
This invention relates to the selection and use of nonpathogenic, lysine-uptake stimulating lactic acid bacteria strains, and products and methods using such strains for example for the improvement of fur and claw quality in pet-animals.
2. Description of the Related Art
In 1908, the Russian biologist Eli Metchnikoff credited the long lives of certain Bulgarian and Russian citizens to the consumption of large amounts of fermented milk products (1). The key organism in these foods was later identified as Lactobacillus acidophilus, a lactic acid-producing bacteria (2). The lactic acid-producing bacteria are so named for their ability to produce lactate. However, lactate production is only one of many benefits derived from this collection of bacteria.
Based on the work of Metchnikoff and others, scientists developed the idea of probiotic microorganisms, directly feeding live, lactic acid-producing bacteria and yeast to animals for improving their health and performance. The observed benefits may result from: 1) competition for attachment sites in the digestive tract, 2) competition for essential nutrients, 3) production of anti-microbial substances, 4) increasing the growth of beneficial bacteria and 5) stimulating the immune system (3).
Some disease-causing bacteria reduce an animal's ability to absorb nutrients by disrupting the lining of the small intestine (4). Studies indicate that the lactic acid-producing bacteria attach to the small intestine and produce substances to prevent disease-causing organisms from binding to the intestinal wall (5). In addition, the attachment of the beneficial bacteria may increase the absorptive surface area of the small intestine and enhance enzyme activity for greater nutrient absorption by the animal (8,6).
Bacteria, both health-promoting and disease-causing, require certain nutrients for growth. Lactic acid-producing bacteria could utilize vitamins, amino acids or other nutrients that might otherwise support the growth of harmful bacteria (7).
Considerable research has focused on the ability of direct-fed microbial cultures to produce substances that inhibit disease-causing organisms. Lactic, acetic and formic acid lower the intestinal pH to create an environment unsuitable for harmful organisms (4). Lactic acid-producing bacteria also secrete hydrogen peroxide, resulting in conditions unfavorable for oxygen-requiring microorganisms (8).
Two groups of antimicrobial substances have been identified, low molecular weight antimicrobial substances, for example, reuterin, produced by L. reuteri; and bacteriocins. Bacteriocins are microbially produced substances that inhibit the growth of bacteria that are often genetically related (4). Bacteriocins are polypeptides and their inhibitory properties are destroyed by proteases while Reuterin, a broad-spectrum antimicrobial substance, is not a polypeptide and its antimicrobial activity is unaffected by proteases.
Research has documented the ability of lactic acid-producing bacteria to inhibit E. coli, Salmonella typhimurium, Staphylococcus aureus and Clostridium perfingens (7). The reduction of diarrhea-causing organisms is especially important in newborn and young animals.
There are however few studies done on probiotics in companion animals. One recent study (9) investigated the application of Lactobacillus acidophilus DSM 13241 in canines. This strain was chosen on the basis of its growth characteristics, antimicrobial activity toward pathogens, and survival rate in gut models. Feeding of healthy dogs resulted in a significant increase in the population of recoverable lactobacilli in the feces with a concomitant decrease in the clostridia population. The researchers concluded that feeding of the probiotic resulted in positive changes in the gut microbiology and in systemic effects that suggested immune system stimulation as observed in humans after they consumed Lactobacillus spp. (10).
Like dogs, cats, mink (Mustela vison) and blue foxes (Alopex lagopus) belong to the mammalian order Carnivora. The carnivores are adapted to relatively concentrated and highly digestible diets, and are characterized by a gastric stomach and a relatively short and uncomplicated intestine. The mink lacks a cecum and has a short digestive tract with very limited bacterial activity in the colon. Dogs and foxes have little cecal capacity and an unsacculated colon, but some bacterial fermentation takes place in the cecum and colon (11).
Protein is vital for growth, repair, and maintenance of body tissue. It forms the basis of enzymes and antibodies. For animals and birds, a lot of protein is used as energy to make fur, feathers and claws, which are primarily alpha-keratin. Alpha-keratins consists of long alpha-helical polypeptides, which are wound around each other to form triple helixes. For fur animals in general, sulfur-containing amino acids are usually considered to be the first limiting ones ((12). Protein to make fur, feathers and claws has to come from the diet. The diets must supply enough amino acids to meet requirements of the essential amino acids with enough excess amino acids to supply nitrogen that makes the nonessential amino acids. Additional protein in the diet cannot be stored and will be converted to fat or excreted by the kidneys.
Lysine is an essential amino acid and thus unable to be synthesized by transamination. It helps make fat usable to the body by producing carnitine, removes toxins from the body, builds fur, feathers and bone. High levels increase the need for arginine. Lysine is highly unstable in intact protein. It reacts with glucose and the reaction accelerates in the presence of warmth and moisture. Research on young pigs and poultry has found lysine to be the first limiting amino acid for growth (e.g. Boisen et al., 2000; (13,14).
Methionine and cysteine are nutrient amino acids, and the most active of the essential amino acids. They are used to make such important molecules as carnitine (used in the transport of fatty acids), creatine, niacin, polyamines, and purines (used to create uric acid). They are also used in the making of fur and feathers. Cysteine can be synthesized from methionine, and therefore it is classified as non essential. However, cysteine and its oxidation product cystine can satisfy approximately 50 percent of the need for total sulfur amino acids and in this way can reduce the need for methionine. Methionine can not be synthesized from cysteine, and therefore it is essential. Methionine can meet the total need for sulfur amino acids in the absence of cystine. Some studies have found methionine to be the first limiting amino acid for hair growth and fur quality in mink. (15,16,17,18,19,20,21)
In most household pets, a healthy skin and coat indicates an animal in general good health. Since skin and coat problems are common in household pets, much research has gone into providing diets which repair deteriorations in skin and coat conditions, thus providing a basic level of healthy skin and coat as described hereinafter.
Research shows that supplementation of a complete and balanced commercial dog food with zinc plus linoleic acid can make significant and substantial enhancements of the skin and coat conditions in dogs. The dogs showed significant improvement in coat gloss and coat scale when compared to control groups receiving a standard diet. This approach is also described in patent EP0987961B1.
Nutrition may impact skin barrier function. The international patent application WO0207531A1 discloses the use of dietary lipids containing anti-microbial fatty acids for the preparation of a food composition intended for improving or maintaining the skin health and/or coat quality.
In the study by Kerminen-Hakkio (21) poor protein quality in mink reduced pelt length on an average by 4 cm. Best overall fur quality was observed in the good quality, high level protein group. The authors conclude that deficiencies in the supply of indispensable amino acids cannot be totally compensated for by increasing the level of protein in the diet.
In some cases, the non-essential amino acids should still be supplemented to ensure optimal available quantity. We have found that available methionine should be balanced with lysine also in food for cats and dogs to improve fur and claw growth and quality. A complimentary way of making better use of lysine from the food, rather than just feeding more, is to look for ways to improve it's absorption.
It is known that animal feeding with Lactic acid bacteria (LAB) is growth-promoting due to generally improved absorption of nutrients. In today's modern animal breeding, a lot of countries already use feed additives which contain probiotic microorganisms for fattening animals. When an animal consumes LAB, they colonize its digestive tract and start to produce enzymes that digest starch (amilase), proteins (proteinases), fat (lipases). These enzymes contribute to the disintegration of complex organic compounds in fodder into low-molecular compounds. The animals can make better use of these low-molecular compounds. Their appetite improves, the fodder conversion rate gets better and the production performances are improved. The use of LAB in animal feeding also contributes to immune defenses against pathogens. LAB disintegrate carbohydrates into lactic acid which reduces pH of the digestive tract content, which become an extremely unfavorable surrounding for pathogen microorganisms.
We have surprisingly found that different strains of LAB present in the food or in the GI-tract can differently aid in the absorption of amino acids, specifically lysine. And that such difference can be measured, thereby making it possible to select the specific strains of lactic acid bacteria that will improve the lysine uptake the most.
The Caco-2 cell system has proved a suitable model system for intestinal epithelial permeability studies (22) of both nutrients, [peptides and amino acids (23,24,25,26)] and pharmaceutical products (27,24) A study performed by Twaites 1996 described the ability of intestinal Caco-2 cells to transport the dibasic amino acid lysine (28).
However there have been few studies on Lactobacillus facilitating or increasing the absorption of amino acids from the diet in pet animals.
Bacterial protein meal (BPM) produced by continuous bacterial fermentation using a defined mixture of four different bacteria (Methylococcus capsulatus, Alcaligenes acidovorans, Bacillus brevis and Bacillus firmus) and natural gas as the carbon and energy source is a novel high-protein feed ingredient. A study was conducted to extend the knowledge of BPM as an ingredient of diets for dogs, using the blue fox as a model animal. The animals revealed generally good growth but there were no data present of fur quality improvement (7).
Patent WO 01/17365A1 describes a method for improving and maintaining the skin and coat system of a pet where the nutritional agent may be a prebiotic or any probiotic microorganism. The applicants mention seventeen different bacterial genera as probiotics and almost thirty specific examples of probiotic microorganisms. Bacillus coagulans Lacris-S strain (SANKYO LIFETECH CO., LTD, Hongo, Bunkyo-ku, Tokyo, Japan) is the only specific strain described and there is no selection of preferred strains mentioned, and nor any based on LAB strains' ability to stimulate lysine uptake.
Although the use of probiotics for better fur and feathering in animals is known in the art, it was not previously known that different probiotic strains varies in their ability to improve the quality of the fur and claws of an animal, for example a dog or a cat, and that this ability is correlated with a probiotic strain's influence on lysine absorption in the gut of the animal.
It is therefore an object of the present invention to select such more suitable strains by analyzing their ability to directly facilitate or improve the absorption of lysine in the animal and to use such a selected strain in animal feed or supplement for example to improving fur and claw quality of a dog or a cat. A new strain Lactobacillus reuteri, ATCC PTA-6127, is shown to stimulate lysine-uptake in fur-animals and this property results in improved fur and claw quality.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.